Genetics for Pseudoalteromonas Provides Tools To Manipulate Marine Bacterial Virus PM2

Abstract

The genetic manipulation of marine double-stranded DNA (dsDNA) bacteriophage PM2 (Corticoviridae) has been limited so far. The isolation of an autonomously replicating DNA element of Pseudoalteromonas haloplanktis TAC125 and construction of a shuttle vector replicating in both Escherichia coli and Pseudoalteromonas enabled us to design a set of conjugative shuttle plasmids encoding tRNA suppressors for amber mutations. Using a host strain carrying a suppressor plasmid allows the introduction and analysis of nonsense mutations in PM2. Here, we describe the isolation and characterization of a suppressor-sensitive PM2 sus2 mutant deficient in the structural protein P10. To infect and replicate, PM2 delivers its 10-kbp genome across the cell envelopes of two gram-negative Pseudoalteromonas species. The events leading to the internalization of the circular supercoiled dsDNA are puzzling. In a poorly understood process that follows receptor recognition, the virion capsid disassembles and the internal membrane fuses with the host outer membrane. While beginning to unravel the mechanism of this process, we found that protein P10 plays an essential role in the host cell penetration.The study of membrane-containing bacteriophages has produced notable knowledge of the assembly and structure of biological membranes and virus structures (18, 32). The availability of genetic tools has been valuable in the study of many bacterial viruses with a membrane (36, 37). The understanding of these sophisticated virus systems has relied on the isolation and analysis of phage mutants. A recent example of this is the X-ray crystallography structure of the membrane-containing phage PRD1, which was solved using a nonsense mutant particle, Sus539, lacking the receptor binding protein P2 (1, 8). Moreover, suppressor-sensitive mutants of PRD1 have been valuable in assigning functions to viral proteins. Recently, we initiated the structural characterization of bacteriophage PM2 as a new model system (2, 19;

Half a Century of Research on Membrane-Containing Bacteriophages: Bringing New Concepts to Modern Virology

“…However, different genome types impose divergent constraints on the DNA injection, presumably giving rise to variation in the viral entry mechanisms. For instance, phage PM2 has been suggested to transfer its circular, supercoiled dsDNA genome into the host by first removing its protein capsid and then fusing its internal membrane with the outer membrane of the Gram-negative host bacterium [51,52,53] (Figure 1c).…”

Genomics of Bacterial and Archaeal Viruses: Dynamics within the Prokaryotic Virosphere

“…Corticoviruses, with their relatively small genomes (ϳ10 kb), might therefore represent an interesting model system where the genetic interplay between different mobile elements (viruses and plasmids) can be investigated. The recently developed genetic system for Pseudoalteromonas-PM2 should facilitate such studies (134). Another important line of research should focus on revealing the role that corticoviruses play in aquatic ecosystems.…”

Clinical isolates ofPseudomonas aeruginosafrom superficial skin infections have different physiological patterns

“…We have studied populations of P. aeruginosa from a common clinical source (diabetic leg ulcers and decubitus wounds) together with well‐defined standard strains and applied established membrane voltage (Daugelavicius et al ., ; Krupovic et al ., , b; Kivela et al ., ) and multiple drug resistance efflux pumps (Daugelavicius et al ., ) monitoring systems. The genetically indistinguishable standard strains overexpressing different Mex pumps resulted in very different phenotypic patterns (Fig.…”